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Generation of insulin-producing cells from the mouse liver using β cell-related gene transfer including Mafa and Mafb.

Nagasaki H, Katsumata T, Oishi H, Tai PH, Sekiguchi Y, Koshida R, Jung Y, Kudo T, Takahashi S - PLoS ONE (2014)

Bottom Line: Furthermore, PDA transfer induced several gene expressions necessary for glucose sensing and insulin secretion in the liver on day 9.However, a glucose tolerance test and liver perfusion experiment did not show glucose-stimulated insulin secretion from intrahepatic β-like cells.They also show that Mafa has a markedly intense and sustained role in β-like cell production in comparison with Mafb.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan.

ABSTRACT
Recent studies on the large Maf transcription factors have shown that Mafb and Mafa have respective and distinctive roles in β-cell development and maturation. However, whether this difference in roles is due to the timing of the gene expression (roughly, expression of Mafb before birth and of Mafa after birth) or to the specific function of each gene is unclear. Our aim was to examine the functional differences between these genes that are closely related to β cells by using an in vivo model of β-like cell generation. We monitored insulin gene transcription by measuring bioluminescence emitted from the liver of insulin promoter-luciferase transgenic (MIP-Luc-VU) mice. Adenoviral gene transfers of Pdx1/Neurod/Mafa (PDA) and Pdx1/Neurod/Mafb (PDB) combinations generated intense luminescence from the liver that lasted for more than 1 week and peaked at 3 days after transduction. The peak signal intensities of PDA and PDB were comparable. However, PDA but not PDB transfer resulted in significant bioluminescence on day 10, suggesting that Mafa has a more sustainable role in insulin gene activation than does Mafb. Both PDA and PDB transfers ameliorated the glucose levels in a streptozotocin (STZ)-induced diabetic model for up to 21 days and 7 days, respectively. Furthermore, PDA transfer induced several gene expressions necessary for glucose sensing and insulin secretion in the liver on day 9. However, a glucose tolerance test and liver perfusion experiment did not show glucose-stimulated insulin secretion from intrahepatic β-like cells. These results demonstrate that bioluminescence imaging in MIP-Luc-VU mice provides a noninvasive means of detecting β-like cells in the liver. They also show that Mafa has a markedly intense and sustained role in β-like cell production in comparison with Mafb.

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Expression of β cell-related genes in mouse liver after Pdx1/Neurod in combination with Mafa (PDA) or Mafb (PDB) gene transfer.Wild-type mouse liver treated with Ad-PDA (PDA, n = 7) displayed a significant increase in β cell-related mRNAs at day 9 (Ins1, Ins2, PC2, i-GK, Sur1, Kir6.2) on Q-PCR analysis. In contrast, the Ad-PDB treated group of mice (PDB, n = 7) displayed a significant increase in Ins1, Ins2, Sur1, and Kir6.2 only at day 3. Data were normalized to Hprt mRNA abundance and shown as relative expression levels to that of the Ad-PDA-treated group at day 3. Data were expressed as the means ± standard errors of the means and analyzed using the Steel-Dwass multiple comparison test.
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pone-0113022-g004: Expression of β cell-related genes in mouse liver after Pdx1/Neurod in combination with Mafa (PDA) or Mafb (PDB) gene transfer.Wild-type mouse liver treated with Ad-PDA (PDA, n = 7) displayed a significant increase in β cell-related mRNAs at day 9 (Ins1, Ins2, PC2, i-GK, Sur1, Kir6.2) on Q-PCR analysis. In contrast, the Ad-PDB treated group of mice (PDB, n = 7) displayed a significant increase in Ins1, Ins2, Sur1, and Kir6.2 only at day 3. Data were normalized to Hprt mRNA abundance and shown as relative expression levels to that of the Ad-PDA-treated group at day 3. Data were expressed as the means ± standard errors of the means and analyzed using the Steel-Dwass multiple comparison test.

Mentions: To examine the gene expression profiles related to β-cell function, we performed quantitative RT-PCR analysis using RNAs recovered from whole liver cells (GFP, n = 6) (Figure 4). The expressions of Pdx1, Neurod, Mafa, and Mafb are shown in Figure S2B. Both Ad-PDA (n = 7) and Ad-PDB (n = 7) treatment induced comparable expression levels of β cell-related genes, including Slc2a2 and Pcsk2, 3 days after infection, but only in the PDA-transfer group were the expressions sustained for longer than 1 week. In contrast to this significant β cell-related gene induction, immunohistochemistry showed that other hormones including glucagon, pancreatic polypeptide (PP), and somatostatin were not induced by the Ad-PDA and Ad-PDB treatments (data not shown). Furthermore, other β cell-specific transcription factors, such as Nkx2.2, Nkx6.2, and Pax4 that are detected in normal β-cell development, were neither induced nor increased by PDA-gene transfer, suggesting that transient β-like cell induction does not occur in the similar molecular cascade to that of the regular developmental process of β cells.


Generation of insulin-producing cells from the mouse liver using β cell-related gene transfer including Mafa and Mafb.

Nagasaki H, Katsumata T, Oishi H, Tai PH, Sekiguchi Y, Koshida R, Jung Y, Kudo T, Takahashi S - PLoS ONE (2014)

Expression of β cell-related genes in mouse liver after Pdx1/Neurod in combination with Mafa (PDA) or Mafb (PDB) gene transfer.Wild-type mouse liver treated with Ad-PDA (PDA, n = 7) displayed a significant increase in β cell-related mRNAs at day 9 (Ins1, Ins2, PC2, i-GK, Sur1, Kir6.2) on Q-PCR analysis. In contrast, the Ad-PDB treated group of mice (PDB, n = 7) displayed a significant increase in Ins1, Ins2, Sur1, and Kir6.2 only at day 3. Data were normalized to Hprt mRNA abundance and shown as relative expression levels to that of the Ad-PDA-treated group at day 3. Data were expressed as the means ± standard errors of the means and analyzed using the Steel-Dwass multiple comparison test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232560&req=5

pone-0113022-g004: Expression of β cell-related genes in mouse liver after Pdx1/Neurod in combination with Mafa (PDA) or Mafb (PDB) gene transfer.Wild-type mouse liver treated with Ad-PDA (PDA, n = 7) displayed a significant increase in β cell-related mRNAs at day 9 (Ins1, Ins2, PC2, i-GK, Sur1, Kir6.2) on Q-PCR analysis. In contrast, the Ad-PDB treated group of mice (PDB, n = 7) displayed a significant increase in Ins1, Ins2, Sur1, and Kir6.2 only at day 3. Data were normalized to Hprt mRNA abundance and shown as relative expression levels to that of the Ad-PDA-treated group at day 3. Data were expressed as the means ± standard errors of the means and analyzed using the Steel-Dwass multiple comparison test.
Mentions: To examine the gene expression profiles related to β-cell function, we performed quantitative RT-PCR analysis using RNAs recovered from whole liver cells (GFP, n = 6) (Figure 4). The expressions of Pdx1, Neurod, Mafa, and Mafb are shown in Figure S2B. Both Ad-PDA (n = 7) and Ad-PDB (n = 7) treatment induced comparable expression levels of β cell-related genes, including Slc2a2 and Pcsk2, 3 days after infection, but only in the PDA-transfer group were the expressions sustained for longer than 1 week. In contrast to this significant β cell-related gene induction, immunohistochemistry showed that other hormones including glucagon, pancreatic polypeptide (PP), and somatostatin were not induced by the Ad-PDA and Ad-PDB treatments (data not shown). Furthermore, other β cell-specific transcription factors, such as Nkx2.2, Nkx6.2, and Pax4 that are detected in normal β-cell development, were neither induced nor increased by PDA-gene transfer, suggesting that transient β-like cell induction does not occur in the similar molecular cascade to that of the regular developmental process of β cells.

Bottom Line: Furthermore, PDA transfer induced several gene expressions necessary for glucose sensing and insulin secretion in the liver on day 9.However, a glucose tolerance test and liver perfusion experiment did not show glucose-stimulated insulin secretion from intrahepatic β-like cells.They also show that Mafa has a markedly intense and sustained role in β-like cell production in comparison with Mafb.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan.

ABSTRACT
Recent studies on the large Maf transcription factors have shown that Mafb and Mafa have respective and distinctive roles in β-cell development and maturation. However, whether this difference in roles is due to the timing of the gene expression (roughly, expression of Mafb before birth and of Mafa after birth) or to the specific function of each gene is unclear. Our aim was to examine the functional differences between these genes that are closely related to β cells by using an in vivo model of β-like cell generation. We monitored insulin gene transcription by measuring bioluminescence emitted from the liver of insulin promoter-luciferase transgenic (MIP-Luc-VU) mice. Adenoviral gene transfers of Pdx1/Neurod/Mafa (PDA) and Pdx1/Neurod/Mafb (PDB) combinations generated intense luminescence from the liver that lasted for more than 1 week and peaked at 3 days after transduction. The peak signal intensities of PDA and PDB were comparable. However, PDA but not PDB transfer resulted in significant bioluminescence on day 10, suggesting that Mafa has a more sustainable role in insulin gene activation than does Mafb. Both PDA and PDB transfers ameliorated the glucose levels in a streptozotocin (STZ)-induced diabetic model for up to 21 days and 7 days, respectively. Furthermore, PDA transfer induced several gene expressions necessary for glucose sensing and insulin secretion in the liver on day 9. However, a glucose tolerance test and liver perfusion experiment did not show glucose-stimulated insulin secretion from intrahepatic β-like cells. These results demonstrate that bioluminescence imaging in MIP-Luc-VU mice provides a noninvasive means of detecting β-like cells in the liver. They also show that Mafa has a markedly intense and sustained role in β-like cell production in comparison with Mafb.

Show MeSH